Features of calmodulin that are important in the activation of the catalytic subunit of phosphorylase kinase

Calmodulin (CaM) is an integral subunit, called δ, of the phosphorylase kinase hexadecamer, and the activity of the isolated catalytic γ-subunit of the kinase is stimulated by CaM. We report here the first analysis of functionally important features of CaM for activation of the γ-subunit. A set of genetically engineered CaMs, in which acidic residues in each of the four E-helices of the "EF-hands" were changed to basic lysine residues, was used to probe the relative importance of charge features in each domain of CaM. The maximal activation of the isolated γ-subunit was diminished by all of the charge reversal mutations. The γ-subunit was especially sensitive to reversals in the second and third E-helix of CaM (residues 45-47 and 82-84), the latter being present in the central helix. The results suggest the functional importance of electrostatics in the interactions between the δ-subunit (CaM) and the catalytic γ-subunit of phosphorylase kinase, which is similar to results obtained with CaM-dependent myosin light chain kinase (MLCK) from chicken gizzard and CaM-dependent protein kinase II (CaMPK-II). However, novel features of the interaction between CaM and the γ-subunit of phosphorylase kinase are the significant contribution of electrostatics throughout the CaM molecule, including residues in both halves and on more than one face of CaM, and the lack of a major effect of the CaM mutations on substrate kinetic parameters, unlike the effects observed with MLCK and CaMPK-II. These results are consistent with a model in which the δ-subunit (CaM) of phosphorylase kinase interacts with an extended region or multiple regions of the γ-subunit and suggest that the mechanism of CaM activation of the γ-subunit may have features that are distinct from those of MLCK and CaMPK-II.